Rotavirus vaccine formulations

ABSTRACT

The present invention provides novel liquid and lyophilized formulations of vaccines against rotavirus infection and methods of their preparation. The formulations include buffering agents appropriate for oral administration of rotavirus vaccines. The formulations also include compounds to stabilize of the vaccine compositions against loss of potency.

This application claims benefit of Provisional Application Ser. No.60/046,760 filed May 16, 1997 also Ser. No. 60/025,754 filed Sep. 26,1996.

CROSS-REFERENCE TO RELATED APPLICATIONS

Not applicable

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

Not applicable

REFERENCE TO A MICROFICHE APPENDIX

Not applicable

FIELD OF THE INVENTION

The present invention is related to novel liquid and lyophilizedformulations of rotaviruses useful as vaccines and methods for theirpreparation.

BACKGROUND OF THE INVENTION

Rotaviruses (RV) cause acute gastroenteritis, a disease that requireshospitalization of infants and young children in developed countries,and a frequent cause of death in children less than 5 years of age indeveloping regions of the world. Studies in the United States,Australia, and Japan have demonstrated that between 34 and 63% ofhospitalizations of children for acute diarrheal disease are associatedwith rotavirus infection. The incidence of hospitalization for rotavirusgastroenteritis in a health maintenance organization in the U.S. wasestimated to be 222 per 100,000 in children from 13 to 24 months of age,and 362 per 100,000 in those less than one year. Infection withrotavirus was associated with 63% of all hospitalizations for acutediarrhea in this pediatric population. A review of mortality data in theU.S. from 1973 to 1983 indicated that 500 deaths per year occur inchildren less than 4 years old due to diarrheal diseases, and that 20 to80% of excess winter deaths due to diarrhea in the U.S. are associatedwith rotavirus infections. Rotaviruses are also responsible forsubstantial proportion of the mortality associated with diarrhealdiseases in third world countries. An effective rotavirus vaccine wouldtherefore have a major impact on the health of children in both thedeveloped and developing areas of the world.

Rotaviruses have an inner and outer capsid with a double-stranded RNAgenome formed by eleven gene segments. Multiple serotypes have beendefined by plaque reduction neutralization tests, and studies ofreassortant viruses have demonstrated that two outer capsid proteins,VP7 and VP4, are the determinants of virus serotype. The VP7 protein iscoded for by either gene segment 7, gene segment 8 or gene segment 9 ofa particular human rotavirus. The location of the VP7 encoding gene maybe determined for each specific rotavirus by conventional experimentalmethods. The VP4 protein is an 88,000 dalton major surface structuralprotein product of gene 4 of a rotavirus. Like VP7, it functions as amajor serotype-specific antigen, operative in serum neutralization (SN)tests, capable of inducing serotype-specific neutralizing antibody, andcapable in a mouse system of inducing serotype-specific immuneprotection against rotavirus disease. In some earlier references, theVP4 was referred to as VP3. After 1988, a change in nomenclature,resulted in the more proper reference to this protein as VP4.

Since the gene segments encoding the VP7 and VP4 proteins segregateindependently, it has been proposed that serotyping nomenclature includeboth the G type, determined by VP7, and the P type, determined by VP4.Most human rotavirus infections in the U.S. are caused by viruses of Gtypes 1, 2, 3, or 4, and P types 1, 2, or 3. However, other humanrotavirus types, including for example, type G9, are more prevalent inAsia, Europe and certain third world countries.

A number of animal rotaviruses are attenuated in humans, and have beenevaluated as potential live rotavirus vaccines, including the bovineserotype G6 WC3 rotavirus. The WC3 vaccine virus was shown to beimmunogenic and non-reactogenic in infants, but was inconsistent inproviding protective immunity against human rotavirus infection. It hasbeen suggested that serotype-specific immunity is necessary to includeconsistent protection against rotavirus diarrhea.

There exists a need to the art for effective vaccines providingprotective immunity against rotavirus infection and the severe clinicalsymptoms associated therewith.

For worldwide distribution of rotavirus vaccines, it is necessary toformulate vaccines such that they are stable under a variety ofenvironmental conditions. Components used to stabilize vaccines areknown. However, particular formulations of components useful tostabilize rotavirus vaccines must be determined experimentally. Oneobject of the present invention is present formulations which stabilizerotavirus vaccines.

SUMMARY OF THE INVENTION

The present invention provides novel formulations of rotaviruses usefulas vaccines and methods for their preparation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Effect of buffer combinations on rotavirus stability at 37° C.for 1 week. Data for the G1 reassortant are shown in panel A and the P1reassortant in panel B. All values are expressed as pfu/mL normalized tothe reference, or 0 day, sample. The buffer combinations are representedas follows: 0.05 M sodium citrate+0.15 M sodium bicarbonate (□), 0.05 Msodium citrate+0.15 M sodium phosphate (◯), 0.05 M lactic acid+0.15 Msodium bicarbonate (Δ), 0.05 M lactic acid+0.15 M sodium phosphate (∇)and 0.20 M sodium succinate+0.05 M sodium phosphate (⋄). Allformulations have pH values of 7.

FIG. 2. Acid neutralizing ability of formulation buffers compared tobicarbonate. One mL of each buffer was titrated with 0.01 N HCl.Symbols: 0.4 M sodium bicarbonate (), 0.1 M sodium citrate+0.3 M sodiumphosphate (◯), 0.1 M sodium citrate+0.3 M sodium bicarbonate (+), and0.2 M sodium succinate+0.1 M sodium phosphate (∇).

FIG. 3. Stability data for reassortant rotavirus in liquid formulationsof 5% sucrose/0.1 M sodium succinate/0.05 M sodium phosphate afterstorage at various temperatures. Data for G1 rotavirus is shown in panelA and for P1 rotavirus in panel B.

FIG. 4. Stability data for reassortant rotavirus in liquid formulationsof 50% sucrose/0.1 M sodium succinate/0.05 M sodium phosphate afterstorage at various temperatures. Data for G1 rotavirus is shown in panelA and for P1 rotavirus in panel B.

FIG. 5. Stability data for G1 rotavirus liquid formulations with higherbuffer, sucrose, and hydrolyzed gelatin concentrations at varioustemperatures. Panel A shows data for G1 rotavirus in Williams' E media("WE"), 50% sucrose, 0.2 M sodium succinate, and 0.1 M sodium phosphate.Stability data for vaccine in Williams' E media, 70% sucrose, 0.2 Msodium succinate, and 0.1 M sodium phosphate is shown in panel B. PanelC shows data for G1 rotavirus in 50% sucrose, 0.1 M sodium citrate, and0.3 M sodium phosphate; panel D shows data for G1 rotavirus in Williams'E media, 50% sucrose, 0.2 M sodium succinate, 0.1 M sodium phosphate,and 5% hydrolyzed gelatin.

FIG. 6. Stability data for P1 rotavirus liquid formulations with higherbuffer, sucrose, and hydrolyzed gelatin concentrations at varioustemperatures. Panel A shows data for P1 rotavirus in Williams' E media,50% sucrose, 0.2 M sodium succinate, and 0.1 M sodium phosphate.Stability data for vaccine in Williams' E media, 70% sucrose, 0.2 Msodium succinate, and 0.1 M sodium phosphate is shown in panel B. PanelC shows data for P1 rotavirus in 50% sucrose, 0.1 M sodium citrate, and0.3 M sodium phosphate; panel D shows data for P1 rotavirus in Williams'E media, 50% sucrose, 0.2 M sodium succinate, 0.1 M sodium phosphate,and 5% hydrolyzed gelatin.

FIG. 7. Stability data for rotavirus liquid formulations in 50% sucrose,0.1 M sodium succinate, and 0.05 M sodium phosphate after storage atvarious temperatures. Data for G2 rotavirus is shown in panel A and forG3 in panel B.

FIG. 8. Stability data for G1 rotavirus lyophilized formulations afterstorage at various temperatures. Panel A shows data for G1 rotavirusdialyzed prior to lyophilization into 1% sucrose, 4% mannitol, and 10 mMsodium phosphate. Stability data for vaccine dialyzed prior tolyophilization into 1% lactose, 4% mannitol, and 10 mM sodium phosphateis shown in panel B. Panel C shows data for G1 rotavirus dilutedphosphate prior to lyophilization into 1% sucrose, 4% mannitol, and 75mM sodium phosphate.

FIG. 9. Stability data for P1 rotavirus lyophilized formulations afterstorage at various temperatures. Panel A shows data for P1 rotavirusdialyzed prior to lyophilization into 1% sucrose, 4% mannitol, and 10 mMsodium phosphate. Stability data for vaccine dialyzed prior tolyophilization into 1% lactose, 4% mannitol, and 10 mM sodium phosphateis shown in panel B. Panel C shows data for P1 rotavirus diluted priorto lyophilization into 1% sucrose, 4% mannitol, and 75 mM sodiumphosphate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel formulations of rotaviruses usefulas vaccines and methods for their preparation. More particularly, theinvention relates to stabilizing formulations for liquid and lyophilizedrotavirus vaccines. In addition, some of these formulations can beorally delivered either with or without preneutralization of gastricacid since some of the formulations contain high levels of bufferingcomponents.

Due to the worldwide distribution of vaccines and the diversity ofambient temperatures, it is necessary to formulate vaccines such thatthey are stable under a variety of environmental conditions. A varietyof stabilization methods have been used. These include the following:

a) Low temperatures (-10° C. to -70° C.). Most vaccines are stableduring storage at extremely low temperatures. However, low temperaturestorage facilities are costly and are not always available; this limitsthe utility and practicality of this approach.

b) Lyophilization. Freeze-dried vaccines are reasonably stable and canbe stored at 2-8° C. for a predefined length of time. Lyophilizationmay, however, result in a loss of viral titer during drying therebyreducing the yield of the manufacturing process. In addition, duringlong-term storage, a lyophilized vaccine may still deteriorate, to thepoint where it may or does not have sufficient titer to conferimmunization. Furthermore, since a lyophilized vaccine requiresreconstitution prior to use, a liquid reconstituted preparation may losepotency while standing at room temperature before use. This loss oftiter during reconstitution may also result in insufficient titer toconfer immunity.

c) Stabilizers. These are specific chemical compounds that interact andstabilize biological molecules and/or general pharmaceutical excipientsthat are added to the vaccine and are used in conjunction with eitherlower temperature storage or lyophilization methods.

These formulations can be prepared by either (1) dilution of bulkvaccine into the stabilizer, (2) dialysis/diafiltration into thestabilizer, or (3) concentration of bulk vaccine and diafiltration intothe stabilizer, followed by lyophilization if required.

The stabilizer composition of the present invention contains thefollowing ingredients in about the amounts indicated. For conveniencethe amounts are stated round numbers. However, one skilled in the artwill recognize that amounts within 10 or 20 percent of the stated valuescan also be expected to be appropriate, i.e., where 20% is stated, arange of from 16-18% to 22-24% is implicit and can be appropriate. Forliquid formulations:

    ______________________________________    Sucrose:               1-70% (w/v)    Sodium or potassium phosphate:                           0.01-2 M    Sodium succinate or sodium citrate:                           0.05-2 M    Tissue culture medium, saline, or water:                           0-balance of                           remaining volume    ______________________________________

For lyophilized formulations:

    ______________________________________    Sodium phosphate         0.05-2 M    Sucrose                  1-20% (w/v)    Mannitol                 1-20% (w/v)    Lactose                  1-20% (w/v)    In addition, the following can also be present:    Hydrolyzed gelatin       2.5% (w/v)    Sodium chloride          150 mM    Sodium glutamate         7 mM    ______________________________________

The following compounds can be used in place of sucrose, and atcomparable osmolality: fucose, trehalose, polyaspartic acid, inositolhexaphosphate (phytic acid), sialic acid or N-acetylneuraminicacid-lactose. Also, any suitable sugar or sugar alcohol such asdextrose, mannitol, lactose, or sorbitol, can be substituted for sucroseat concentrations effective in achieving the desired stabilization.

The concentration of sugar relates to the viscosity of the formulation.In instances where reduced viscosity is desired, it is known in the artto be preferable to use lower concentrations of sugar, e.g., sucrose. Itwill also be appreciated by persons in the art that the upper limit forthe concentration of sugar can be dictated by the ability of aformulation to undergo required filtration or processing steps.

Tissue culture medium, saline or water can be used as a diluent.Frequently, Williams' E medium ("WE") is used, by which we mean eitherWilliams' E medium or Williams' medium E modified.

Also, buffering agents to neutralize gastric acid are not limited tocitrate, phosphate and succinate and could include bicarbonate or commoncarboxylic acids (carboxylates) such as, but not limited to, fumarate,tartarate, lactate, maleate, etc. The appropriateness of any of thesecan be assessed by simply trying a formulation in which these agents aresubstituted or combined with phosphate, citrate or succinate. Up toabout 2.0 M carboxylates can be used in the liquid and lyophilizedformulations of this invention, however, we prefer to use less thanabout 1.0 M, e.g., about 0.05-0.9 M, and can be less than about 0.7 M,e.g., 0.05 to about 0.7 M. It is also preferable to use less than 0.5 M,e.g., about 0.05 to 0.45 M. Particular concentrations in these rangescan be appropriate. Also, higher concentrations of buffering components(e.g. phosphate, succinate, citrate) can be used if, for example,additional gastric neutralization is required. In instances whereadditional buffering capacity is useful in phosphate/citrate orphosphate/succinate buffers, it is preferable to further increase theconcentrations of succinate or citrate as the buffering agent ratherthan phosphates.

Up to about 2.0 M phosphate can be used in the liquid and lyophilizedformulations of this invention, however, we prefer to use less thanabout 1.0 M, e.g., about 0.010-0.8 M, and often less than 0.5 M, e.g.,about 0.010 to 0.45 M. It is most preferable to use less than about 0.35M, e.g., 0.010-0.30 M. Particular concentrations in these ranges can beappropriate. In liquid formulations, we prefer to maintain theconcentration of phosphate about or below 0.30 M, e.g., 0.010-0.35 M toavoid the precipitation of phosphate salts, e.g., during long termstorage or freeze/thaw cycles. Thus, the upper limit for theconcentration of phosphate in any particular formulation can be dictatedby the formation or precipitation of phosphate salts and whether thesalts negatively affect the performance of the formulation in areas suchas stablility and administration. Particular concentrations can bereadily determined for any particular formulation by standard empiricaltesting including pH adjustments in the range of pH 6-8.

For general guidance, examples of the acid neutralizing capacities ofsome liquid formulations are presented in Table 1 below. Also providedare some preferred formulations.

                  TABLE 1    ______________________________________    Acid-neutralizing capacities (ANC) of rotavirus stabilizer formulations.    Sodium Phosphate                 Sodium Citrate                              Sucrose ANC    (M)          (M)          (%)     (mEq/mL)    ______________________________________    0.30         0.10         50      0.48    0.30         0.70         50      1.55    0.75         0.25         50      1.07    For lyophilized formulations:    Sodium phosphate     20 mM    Hydrolyzed gelatin   2.5% (w/v)    Sucrose              5% (w/v)    Sodium chloride      150 mM    Sodium glutamate     7 mM    or    Sucrose or Lactose   1% (w/v)    Mannitol             4% (w/v)    Sodium or potassium phosphate                         0.01-0.1 M    A preferred formulation of the liquid viral vaccine stabilizer    of the present invention is as follows:    Sucrose              50% (w/v)    Sodium or potassium phosphate                         0.1 M    Sodium succinate     0.2 M    Tissue culture medium                         used for all dilutions    or    Sucrose              50% (w/v)    Sodium or potassium phosphate                         0.3 M    Sodium citrate       0.1 M    Tissue culture medium                         used for all dilutions    Sucrose              30% (w/v)    Sodium or potassium phosphate                         0.3 M    Sodium citrate       0.7 M    Tissue culture medium                         used for all dilutions    ______________________________________

In these preferred formulations, it can be appropriate to use saline orwater in place of, or in combination with, the tissue culture medium.

This invention involves formulations of reassortant rotaviruses (RRV)suitable for use as vaccines, which are characterized by safety tohumans and the ability to confer immune protection against humanrotavirus infection. The RRV are produced by genetic reassortmentbetween an attenuated bovine rotavirus (preferably WC3 or progenythereof) and at least one rotavirus representing an epidemiologicallyimportant human serotype. In one type of RRV, the human rotaviruscontributes to the reassortant at least the gene segment encoding theVP7 protein. In another type of RRV, the human rotavirus parentcontributes to the reassortant at least the gene segment encoding theVP4 protein. In still another type of RRV, the human rotavirus parentalstrain contributes at least both the VP7 and VP4 gene segments. Inadditional types of RRV, the human rotavirus parental strain maycontribute gene segments in addition to those which encode the VP7and/or VP4 antigens.

The human rotavirus gene which encodes for the neutralization antigenVP7 and/or VP4 in the RRV may be selected from any human rotavirusserotype for which immunization is desired. Desirably, in a reassortantof this invention the VP7 gene is derived from a G1, G2, G3, or G4 humanrotavirus serotype and the VP4 protein is derived from a human P1 or P2serotype. Among the rotavirus strains noted to be clinically significantin human rotavirus infections (hereinafter "human rotavirus strains"),including strains useful in the present invention, are the strainsprovided below:

serotype G1: WI79, Wa, D;

serotype G2: strains WISC2 and DS1;

serotype G3: strains WI78, P, HCR3A;

serotype G4: Bricout (Br) B, ST3;

serotype G8: 69 M;

serotype G9: WI61;

serotype P1: WI79, WI78, WI61, Wa;

serotype P2: DS1; and

serotype P3: WISC2, BrB, BrA, M37.

This list of human rotavirus strains is non-exclusive. For example,several rotavirus strains previously identified in animal infectionshave also been found in human infections. These strains can beanticipated to be useful as `human` rotavirus strains for the purposesof this invention, e.g., the `porcine` rotavirus OSU, a serotype G5, andthe `bovine` rotavirus B223, a serotype G10. One of skill in the art canreadily obtain other appropriate human strains from suitabledepositories or academic or commercial sources.

The non-human genes present in the reassortants of this invention areobtained preferably from the attenuated, serotype G6, bovine rotavirusstrain WC3 or its progeny, described in detail in U.S. Pat. No.4,636,385. However, other rotavirus reassortants, particularly otherbovine reassortants, are also preferred.

                  TABLE 2    ______________________________________    Human     Parent or    Serotype  Reassortant   ATCC#    Deposit Date    ______________________________________    G1        WI79-3,9.sup.a                            VR2194   Nov. 25, 1987                            VR2196   Nov. 25, 1987              WI79-4,9      VR2415   July 8, 1993    G2        WI79-3 + WISC2-9       Dec. 7, 1994              WISC2 parental                            VR2417   July 8, 1993              strain    G3        WI78-8                 Dec. 7, 1994              WI78-1,6-11   VR2195   Nov. 25, 1987              WI78-1,7-11.sup.b    G4        Bricout B-9            Dec. 7, 1994    P1        WI79-4        VR2377   June 19, 1992              WI79-4,9      VR2415   July 8, 1993              WI61-4.sup.b    P2        DS1-4.sup.b    ______________________________________     .sup.a Originally named WI799. The two deposits represent different     passage levels of the reassortant.     .sup.b Not deposited.

The deposits of WI79-3,9 and WI78-1,6-11 have been converted to complywith requirements of the Budapest Treaty. All other deposits have beenoriginally made under the Budapest Treaty. All restrictions on theavailability to the public of the deposited material identified in Table2 will be irrevocably removed upon the grant of a patent on thisapplication, the culture(s) will be maintained for a period of 30 yearsfrom the deposit date, or at least five years after the most recentrequest for a sample, whichever is longer; and the deposit will bereplaced if viable samples cannot be dispensed by the depository. Duringthe pendency of this patent application, access to these deposits willbe afforded to one determined by the Commissioner to be entitledthereto.

Vaccine Compositions

Vaccines for providing immunological protection against acute diarrheacaused by human rotavirus infection can contain one or more rotavirusreassortants in a formulation of the present invention. Exemplaryrotavirus reassortants and combinations thereof and their use invaccines are found in U.S. Pat. No. 5,626,851 and in U.S. applicationSer. No. 08/456,906, both of which are incorporated herein by referencesin their entireties. Several exemplary vaccine compositions aresummarized in Table 3.

                  TABLE 3    ______________________________________    Vaccine compositions                     Preferred Reassortants    ______________________________________    G1 + G2 + G3 + G4                     WI79-3,9 + (WI79-3 + WISC2) +                     WI78-8 + BrB-9    G1 + G2 + G3 + G4 + P1                     WI79-3,9 + (WI79-3 + WISC2-9) +                     WI78-8 + BrB-9 + WI79-4    G1 + G2 + G3 + P1                     WI79-3,9 + (WI79-3 + WISC2-9) +                     WI78-8 + WI79-4    G1 + P1          WI79-3,9 + WI79-4    G1 + G2 + G3     WI79-3,9 + (WI79-3 + WISC2-9) +                     WI78-8    G1 + G2 + G3 + G4 +                     WI79-3,9 + (WI79-3 + WISC2-9) +    P1 + P2          WI78-8 + BrB-9 + WI79-4 + DS1-4    G1               WI79-3,9    ______________________________________

The rotavirus vaccines of the invention can contain conventionalcomponents. Suitable components are known to those of skill in the art.These vaccine compositions can be prepared in liquid forms or inlyophilized forms. Other optional components, e.g., stabilizers,buffers, preservatives, flavorings, excipients and the like, can beadded. The determination of specific formulations useful in stabilizingvaccine compositions has required extensive experimentation.

When adapted for oral administration, one formulation includes as acarrier Williams' E medium ("WE")/50% sucrose/0.1 M succinate/50 mMphosphate liquid. Other formulations include 0.2 M succinate and 0.1 Mphosphate, or 0.1 M citrate and 0.3 M phosphate. Another formulationincludes 0.7 M citrate and 0.3 M phosphate with Williams' E medium/30%sucrose. In addition, novel adjuvants to boost or augment immuneresponses developed for oral administration should be compatible withthese formulations. When adapted for parenteral administration,conventional adjuvants (e.g., aluminum salts) or novel adjuvants canalso be employed in the vaccine composition.

Optionally, the vaccine may preferably be formulated to contain otheractive ingredients and/or immunizing antigens. For example, when adaptedfor oral administration, formulation with the Sabin polio vaccine may bedesirable.

The dosage regimen involved in a method for vaccination, including thetiming, number and amounts of booster vaccines, will be determinedconsidering various hosts and environmental factors, e.g., the age ofthe patients time of administration and the geographical location andenvironment.

Method of Vaccination

Therefore, also included in the invention is a method of vaccinatinghumans against human rotavirus infection with the novel RRV vaccinecompositions. The vaccine compositions including one or more of thereassortants described herein are administered, preferably by the oralroute, in a suitable dose, preferably liquid.

The dosage for all routes of administration is generally between 10⁵ and10⁹ plaque forming units (pfu) of the reassortant with the preferreddosage being 10⁷ pfu. Additional doses of the vaccines can be also beadministered. It may be preferable to inoculate susceptible infants andchildren on an annual basis prior to the "rotavirus season". Rotavirusinfection in humans has been observed to occur in various geographicalregions during the same season, e.g., in winter in the United States.Repeated inoculations prior to that season for susceptible infants andchildren may be indicated. For example, one currently preferred dosageregimen for the U.S. includes three doses approximately two months apartprior to the beginning of the rotavirus season.

The following examples illustrate methods for preparing the RRV vaccineformulations of the invention. These examples are illustrative only anddo not limit the scope of the invention.

EXAMPLE 1

Administration of a vaccine by the oral route exposes the vaccine to thelow pH gastric environment. Most vaccines tend to be inactivated by suchextreme conditions. In order to ensure delivery of active vaccine,potential buffers were examined for acid neutralizing capacity as wellas their ability to stabilize rotavirus.

Rotavirus Stability in the Presence of Acid Neutralizing Buffers

Citrate, lactate, and succinate buffer combinations (5 total) wereevaluated for their effect on rotavirus stability at 37° C. over a 1week period. The buffers, whose concentrations are given in the legendto FIG. 1, were added to an equal volume of rotavirus in WE medium andincubated for 0, 3, or 7 days.

For the G1 serotype, the bicarbonate combinations had no effect on thetime to lose one half of the infectious titer (t_(1/2)) since the valueswere similar to those in 5% sucrose (0.5 days). In contrast, thephosphate buffers containing citrate, lactate, and succinate stablilizedthe virus exhibiting t_(1/2) values of 1.2, 1.4, and 1.5 days,respectively (FIG. 1).

As shown in FIG. 1, phosphate had a similar effect on the stability ofP1. The lactate/phosphate buffer had a t_(1/2) of 2.4 days, and thesuccinate/phosphate combination had a t_(1/2) of 6.8 days compared to avalue of ca. 1.2 days for a 5% sucrose solution. Similar to their effecton the G1 rotavirus, the buffer combinations containing bicarbonateconferred less stability on the P1 serotype than similar bufferscontaining phosphate.

Combination of Rotavirus with Acid Neutralizing Buffer--Potential SingleAdministration

The stabilizing effect of succinate/phosphate as well as other bufferssuggests that the formulation can contain an acid neutralizer. One mL ofthe buffers tested appear to neutralize enough acid to keep the pH above3.5 (FIG. 2) which is known from our direct experimentation and thescientific literature to be necessary for preservation of rotavirusinfectivity. Based on infant gastric acid volumes and acid secretionrates, the pH can be maintained in vivo for approximately 0.5 h with theliquid formulations to be described in this work, however, humanclinical studies will have to be performed to confirm these assumptions.As another test of buffering ability, the USP test for acid-neutralizingcapacity was performed. As shown in Table 4, RV formulation bufferingcomponents are more effective than an equal volume of infant formula.

                  TABLE 4    ______________________________________    Acid-neutralizing capacity as measured by USP    test for a novel liquid rotavirus formulation (1); formulation used in    previous clinical trials by others (2); tissue culture media (3); infant    formula (4), and an antacid (5).                         mEq/mL    ______________________________________    (1)   50% sucrose + 0.2 M sodium succinate                               0.41          + 0.1 M sodium phosphate in          Williams' E media ("WE")    (2)   0.3 M sodium bicarbonate                               0.40          + 0.033 M sodium citrate    (3)   Williams' E media    0.02    (4)   Isomil ®         0.12    (5)   Mylanta ®        5.17    ______________________________________

For lyophilized formulations, additional buffering capacity can beattained by reconstitution with an acid-neutralizing buffer described inthis work or commonly available acid neutralizing compounds such as abicarbonate solution. Thus, with either a liquid or lyophilizedformulation, adequate buffering capacity is possible withoutpretreatment. Consequently, the rotavirus vaccine may preferably be ableto be administered in a single administration rather than with aseparate gastric neutralization step followed by the vaccine. However,simultaneous administration of buffer and vaccine will have to befurther evaluated in patients to have confidence in the efficacy of thevaccine using in this approach. If pretreatment of patients (formulafeeding or dose of bicarbonate or an antacid such as Mylanta®) is stillnecessary to ensure adequate gastric acid neutralization for routineoral vaccination with rotavirus, these formulations will still provide alarge enhancement in the storage stability as described in the nextsection. Furthermore, the rotavirus reassortants are compatible withinfant formulae (e.g., Isomil® and Similac®) as well as bicarbonatebuffers and show comparable thermal stability in the presence or absenceof these neutralizers.

EXAMPLE 2

Putative binding sites on rotavirus can be considered as targets forstabilization. Calcium and zinc binding sites have been suggested to bepresent in rotavirus proteins and the presence of these cations maystabilize the vaccine. Other divalent cations may also bind to these orother sites and stabilize rotavirus and its reassortants. Binding byother compounds was also investigated in order to identify compoundsthat can stabilize the vaccine yet not interfere with its ability toconfer immunogenicity.

a. Effect of Divalent Metal Ions

The addition of metal chelators such as EDTA or EGTA is known to cause aloss in RV infectivity, presumably by disrupting the outer shell of theRV. This suggests that metals may be necessary for structural integrity.Accordingly, divalent metal ions were examined to assess their potentialability to stabilize rotavirus (RV).

Rotavirus in WE medium was dialyzed at 4° C. for approximately 16 hoursin 20 mM Tris buffer and 100 mM NaCl. The final solution wassupplemented with 10 mM of either CaCl₂, MnCl₂, MgCl₂, ZnCl₂, or CaCl₂+ZnCl₂ to yield a final concentration of 10 mM metal ion. The samplescan be filtered prior to formulation. Samples were incubated at 37° C.for 0, 2/3, and 7 days and were then stored at -70° C. until assayed.Each data point represents an average of 2 replicate samples.

As shown in Table 5, calcium and manganese do improve the stability ofboth G1 and P1 rotavirus reassortants at 37° C. when the formulationsare prepared by dialysis of the rotavirus bulks into formulationswithout tissue culture medium. Zinc dramatically decreased theinactivation half-life (t_(1/2)) of G1 and significantly decrease thet_(1/2) of P1 in the presence or absence of calcium. It is possible thatZn²⁺ may be replacing Ca²⁺, causing the destabilization of the outercapsid in a manner analogous to the removal of Ca2+ by EDTA. Analternative explanations may be that Zn²⁺ activates endogenousmetalloproteinases or potentiates nucleases derived from the cellculture. The addition of divalent metals does not increase the thermalstability of RV when formulated in a stabilizer containing tissueculture medium such as Williams' E or Williams' modified E. The G2 andG3 reassortants appeared to behave similarly to G1 and P1 reassortantswhen compared in cation-supplemented tissue culture media.

Thus, in preparing stabilized formulations of rotaviruses as describedherein, it is preferrable that sufficient levels of divalent metal ionsbe present. These metal ions are most likely provided by the tissueculture medium and cells used in cell culture to prepare the bulk virus.Metal ions can also be supplemented, if necessary, in the finalformulation by direct addition individually or through the use of tissueculture medium.

                  TABLE 5    ______________________________________    Effect of divalent metals on the inactivation kinetics    of rotavirus reassortants. Values represent the log loss in viral titer    after 3 days at 37° C.    Cation (10 mM) added                       P1     G1    ______________________________________    none               2.2    2.5    Ca.sup.2+          0.5    0.2    Zn.sup.2+          >3.8   >4.0    Zn.sup.2+  + Ca.sup.2+                       >3.9   >3.9    Mn.sup.2+          1.5    2.2    Mg.sup.2+          2.6    4.2    ______________________________________

b. Effect of Biologically Relevant Sugars and Polyanions

Preliminary experiments described above showed rotavirus reassortantsare stabilized by phosphate buffer. Since there are examples ofmonomeric proteins which are stabilized by phosphate that are alsostabilized by related polyanions such as sulfate, inositol hexaphosphate(phytic acid) and various sulfated compounds (heparin and sulfatedβ-cyclodextrin), these compounds were tested for their ability tostabilize rotavirus. Polymeric forms of polyanions are generally moreeffective stabilizers since a higher charge density can be maintained atlower concentrations of ligand, therefore, polyaspartic acid was alsoexamined due to its high negative charge density. Sialic acid(N-acetylneuraminic acid) was examined since it may bind to VP4 and,therefore, may provide protection from thermally-induced degradation orunfolding. Likewise, sialic acid derivatives such as N-acetylneuraminicacid-lactose and mucin were tested. The loss of RV infectivity with hostmaturation has been suggested to be due to a switch in the presence ofsialic acid to fucose; consequently fucose was examined. Lastly,trehalose was examined because of its reputed properties as a favorabledrying excipient.

As can be seen in Table 6, a variety of compounds can be added torotavirus formulations and stabilize the virus during acceleratedstability testing. Inositol hexaphosphate showed the greatest ability tostabilize RV compared to the other ligands in this study. For G1, a4-fold increase in thermal stability at 37° C. was observed. Mucinprevents infectivity, probably not by destabilizing the virion structurebut rather sequestering RV (clumps were observed prior to assay). Thesulfated polymers had a negligible effect, however, all other testedcompounds stabilized RV to varying degrees. For example, trehaloseextended the inactivation half-life for G1 by greater than 2-fold and P1by less than 50%.

Sialic acid stabilized both G1 and P1 RV. Sialic acid should stabilizethe G types and not the P types if the binding site is located on VP4.In these experiments, P1 appeared to have a lower half-life in thepresence of polyanions in general. The lower t_(1/2) in the presence ofheparin and poly aspartic acid may suggest that RV bind more tightly tothese ligands rather than being destabilized by them. The mechanism ofstability suppression is not entirely clear. A low level of infectivityas measured by the plaque assay can be caused by destabilization of thevirion itself or sequestration of RV by the ligand. If the associationbetween RV and the excipient is moderate, the ligand would be expectedto dissociate under the diluted conditions of the assay (as well as invivo). Tightly bound complexes can contain stable viral particles, yetare not infectious since they are unable to dissociate. This latter caseappears to apply to mucin, heparin, and possibly polyaspartic acid.Also, adverse effects of the excipients on the cells used in the plaqueassay cannot be disregarded. Regardless of the mechanism, certainpolyanions provide no advantage. Inositol hexaphosphate appears to bethe most favorable of all the ligands examined, exceeding the stabilityinduced by phosphate-containing buffers. These results also supportprevious studies described in this work which show phosphatedramatically stabilizes RV. Thus, a variety of phosphates (e.g.,monophosphates and polyphosphates) and phosphorylated compounds (e.g.,phosphorylated sugars) can stabilize rotavirus.

                  TABLE 6    ______________________________________    Effect of polyanions and sugars on the    inactivation kinetics. Samples were incubated at 37° C. for 1    week.                       t.sub.1/2 (days) for                                  t.sub.1/2 (days) for    added to RV in WE  G1         P1    ______________________________________    5% sulfated β-cyclodextrin                       0.5        0.8    5% fucose          1.2        1.7    5% poly-aspartic acid                       1.5        0.6    1% inositol hexaphosphate                       2.0        3.2    1% heparin         0.7        <0.1    1% sialic acid     0.8        1.4    1% N-acetylneuraminic acid-lactose                       1.2        1.5    1% mucin           <0.1       <0.1    5% trehalose       1.3        2.0    5% sucrose         0.5        1.4    ______________________________________

EXAMPLE 3

One-year probe stability data were obtained for several optimizedlyophilized and liquid formulations of G1 and P1 rotavirus at varioustemperatures and compared to the stability data of an unoptimizedformulation, WE medium/5% sucrose. Optimized liquid formulationscontaining rotavirus reassortants in WE medium containing sucrose,sodium phosphate, and sodium succinate or sodium citrate showed asubstantial improvement in stability. Further improvements in storagestability were observed for lyophilized formulations. With theappropriate formulation, the thermostability of rotavirus exceeds thatof existing live-virus liquid (i.e., OPV) and lyophilized (e.g.,measles) vaccines.

The stabilizing effect of either the succinate/phosphate or thecitrate/phosphate buffers offers the potential of combining stabilityenhancement with a gastric neutralization. Liquid formulations as wellas lyophilized formulations that can be reconstituted using this buffercan allow the formulation to be delivered in a single administration.

a. Liquid Formulation Stability Data

When formulated in Williams' E medium/5% sucrose/0.1 M succinate/50 mMphosphate at pH 7, the G1 rotavirus reassortant vaccine loses 0.7 logtiter after 1 year at 4° C. when compared to samples stored at -70° C.(FIG. 3). The P1 reassortant vaccine loses 0.2 log under the sameconditions. After 6 months at 22° C., the G1 reassortant lost 2.6 logsof infectious titer while the P1 reassortant rotavirus lost 5.2 logs.This can be compared to the unoptimized liquid formulation of the G1reassortant in Williams' E medium/5% sucrose that was recently used inclinical trials which lost greater than 5 logs of infectivity afterincubation for 6 months at 22° C. and 1-2 logs at 4° C. after one year.These data demonstrate the additional stabilizing effect of the specificbuffer combinations described in this work.

In Williams' E medium/50% sucrose/0.1 M succinate/50 mM phosphate at pH7, the G1 rotavirus reassortant vaccine loses 0.8 logs titer after 1year at 4° C. when compared to samples stored at -70° C. (FIG. 4). TheP1 reassortant vaccine loses less than 0.3 logs under the sameconditions. At 22° C., both G1 and P1 vaccines lose about 2 logs ofinfectivity after 1 year. These data demonstrate the additionalstabilizing effect of high sugar concentrations.

Additional formulations with higher buffer concentrations (Williams' Emedium/50% sucrose/0.2 M succinate/0.1 M phosphate, pH 7) furtherstabilize the G1 rotavirus vaccine at 4° C. resulting in no significantloss of titer when compared to similar samples stored at -70° C. (FIG.5). Moreover, no loss in G1 titer is observed for any of the optimizedliquid formulations stored at 4° C. for one year. The infectivity of theP1 reassortant is 0.2 logs less than the -70° C. samples for allformulations (FIG. 6). Although the stabilities of both G1 and P1rotavirus reassortants at 4° C. are similar for formulations usinghigher buffer concentrations, the formulation containing Williams' Emedium/50% sucrose/0.1 M citrate/0.3 M phosphate at pH 7 shows less lossat 22° C. when compared to other formulations. For example, G1 rotavirusin Williams' E medium/50% sucrose/0.2 M succinate/0.1 M phosphate showsa 1.5 log loss in titer after one year at 22° C., whereas the Williams'E medium/50% sucrose/0.1 M citrate/0.3 M phosphate formulation showsonly a 0.6 log loss after this period. The higher phosphateconcentration in the latter formulation can be responsible for theincreased stability since the presence of phosphate and phosphorylatedcompounds increase the thermostability of rotavirus reassortants asdemonstrated by our earlier screening experiments. Although rotavirus inthe citrate/phosphate buffered formulation appears to be more stable at22° C., it is less stable at 45° C. for both reassortants and at 37° C.for P1 rotavirus.

After 12 months at 4° C. in Williams' E medium/50% sucrose/0.1 Msuccinate/50 mM phosphate at pH 7, the G2 rotavirus reassortant lost 0.2log of infectivity and the G3 reassortant decreased in titer by 0.3 logwhen compared to similar samples stored at -70° C. (FIG. 7). Compared toG1 and P1 reassortants in similar formulations (FIG. 3), G2 and G3 havestabilities comparable to that of the P1 rotavirus reassortant andbetter than that seen with the G1 reassortant at 4° C. However, the G2and G3 vaccines appear to be less stable than the G1 vaccine at 22° C.

The stability of G1 reassortants was studied in the presence and absenceof tissue culture medium in formulations including sucrose, phosphateand citrate (Table 7). Formulation A, containing only 5% sucrose in WE,served as the standard in this study. Test formulation B contains 0.3 Msodium phosphate and 0.1 M sodium citrate with 50% sucrose in WE. Testformulation C contains 50% sucrose, 0.3 M sodium phosphate and 0.1 Msodium citrate without WE. The viral bulk is diluted 10-fold intoformulations B or C ,. Thus, 100% of the liquid medium in B is tissueculture medium whereas 10% of the liquid medium in C is tissue culturemedium. In C, the viral bulk is the only source of tissue culturemedium. As shown in Table 7, formulations B and C showed greaterstability that formulation A. The presence or absence of tissue culturemedium in the formulations had a small, but measureable, effect on thestability of the rotavirus at 30° C. (compare B and C, Table 7). Thiseffect was greater at 37° C. but still small compared to the standard(Formulation A). These data indicate that a wide concentration range(10-100%) of tissue culture medium is acceptable to attain improvedstability.

                  TABLE 7    ______________________________________    Potency loss (as log pfu/mL) of G1 rotavirus using formulations    with and without tissue culture medium.                    A           B     C    ______________________________________    Loss after 1 week at 30° C.                    3.2         0.7   0.6    Loss after 1 week at 37° C.                    >6.5        0.6   1.0    ______________________________________

To examine the effect of tissue culture medium at volume proportions ofless than 10%, dialysis was employed to completely remove the tissueculture medium from the virus bulk. When a rotavirus liquid formulationwas prepared from dialyzed virus bulk and thus contained 0% tissueculture media in the final formulation, these preparations inactivatedfaster than preparations in which rotavirus bulk was simply diluted intoa stabilizer without tissue culture media (resulting in 10% tissueculture medium being present in the final vaccine formulation). Thissuggests that dialysis may have removed essential stabilizing componentsthat are present in WE tissue culture medium. In the absence of aneffective amount of tissue culture medium, divalent cations such ascalcium can be added to the dialyzed vaccine formulation to improvestability (see Table 5). Dialysis at various processing scales can alsobe performed using diafiltration or ultrafiltration methods.

The stability of G1 reassortants was studied over a range of pH.Rotavirus G1 reassortant was formulated in 0.3 M sodium phosphate/0.1 Msodium citrate/50% sucrose stabilizer at different pH values. The viraltiter indicates that under accelerated stability conditions, thestability of G1 reassortant is greater in the range from about pH 4.0 toabout pH 8.0, particularly between about pH 5.0 to about pH 7.0. By"about pH" we mean within approximately 0.3 units of the stated pHvalue.

                  TABLE 8    ______________________________________    Potency log loss of G1 rotavirus after 1 month at 30 or 37° C. in    0.3 M    sodium phosphate/0.1 M sodium citrate/50% sucrose stabilizer    at various pH values.             1 month at 30° C.                       1 month at 37° C.    ______________________________________    pH 3       4.6         >6    pH 4       1.3         >6    pH 5       1.3         1.5    pH 6       1.3         1.4    pH 7       1.4         2.2    pH 8       1.6         >6    ______________________________________

b. Lyophilized Formulation Stability Data

The G1 vaccine showed a 0.3 log loss after one year at 22° C. in alyophilized formulation of 1% sucrose/4% mannitol/10 mM sodium phosphateat pH 7 (FIG. 8). Formulations containing 1% sucrose/4% mannitol/75 mMsodium phosphate at pH 7 showed no significant losses after one year attemperatures of 22° C. or below. P1 vaccines showed lower stability thanthe corresponding G1 formulations. In 1% sucrose/4% mannitol/10 mMsodium phosphate at 4° C. for one year, the P1 reassortant shows a 0.4log loss in titer when compared with the vaccine stored at minus 70° C.(FIG. 9). A similar formulation with higher phosphate shows a loss ininfectivity of less than 0.2 logs. The P1 vaccine in a phosphate,sucrose and hydrolyzed gelatin stabilizer shows no significant lossafter one year at 4° C. These lyophilized formulations were preparedeither by 10-fold dilution of rotavirus bulk into stabilizer (finalconcentration of 10% tissue culture medium) by dialysis of rotavirusbulk into stabilizer (complete removal of tissue culture medium).

What is claimed is:
 1. A liquid rotavirus vaccine formulationcomprising:a) at least one strain of rotavirus about 1×10⁵ to about1000×10⁵ pfu/mL; b) sugar about 1 to about 70% (w/v); c) phosphate about0.01 to about 2 M; and d) at least one carboxylate about 0.05 to about 2M.
 2. The formulation of claim 1 wherein said at least one carboxylateis selected from the group consisting of succinate, citrate, fumarate,tartarate, maleate and lactate.
 3. The formulation according to claim 1wherein said sugar is selected from the group consisting of sucrose,mannitol, lactose, sorbitol, dextrose, fucose, trehalose, polyasparticacid, inositol hexaphosphate (phytic acid), sialic acid orN-acetylneuraminic acid-lactose.
 4. The liquid vaccine formulation ofclaim 1 further comprising:e) at least one diluent selected from thegroup consisting of tissue culture medium, saline and water.
 5. Theformulation of claim 1 wherein the concentration of sugar is betweenabout 5 to about 70%; the concentration of phosphate is between about0.05 to about 0.3 M; and said at least one carboxylic acid is citrate orsuccinate at a concentration between about 0.05 to about 0.7 M.
 6. Theformulation according to claim 1 wherein the pH is between about pH 5.0to about pH 8.0.
 7. The formulation according to claim 1 wherein saidphosphate is selected from the group consisting of monophosphates,polyphosphates and phosphorylated compounds.
 8. The formulationaccording to claim 7 wherein said phosphorylated compounds arephosphorylated sugars.
 9. A lyophilized rotavirus vaccine formulationcomprising:a) at least one strain of rotavirus about 1 to about 1000×10⁵pfu/mL); b) at least one sugar about 1 to about 20% (w/v); and c)phosphate about 0.05 to about 2 M.
 10. The formulation according toclaim 9 wherein said at least one sugar is selected from the groupconsisting of sucrose, mannitol and lactose.
 11. The formulationaccording to claim 9 wherein upon reconstitution with diluent the pH isbetween about pH 5.0 to about pH 7.0.
 12. A method of preparingstabilized rotavirus vaccine formulations according to claim 1,comprising:(a) cultivating a rotavirus and mixing the rotavirus with aconcentrated stabilizing solution to form a virus bulk; and, optionally,(b) dialyzing the virus bulk to form a stabilized rotavirus vaccinesolution of claim
 1. 13. A method of administering an oral rotavirusvaccine formulation to an individual comprising treatment of theindividual oral rotavirus vaccine formulation having sufficientbuffering capacity to neutralize stomach acid.
 14. The formulation ofclaim 9 comprisinga) at least one sugar selected from the groupconsisting of sucrose and lactose about 1% (w/v); b) mannitol about 4%(w/v); and c) phosphate about 0.010 to about 0.075 M.